White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. They are also involved in allergies/asthma due to the development of an unwanted or excessive type of immune response to substances (antigens) in our environment and in autoimmune diseases. Because T cells respond to foreign substances (antigens) in the form of peptide- major histocompatibility complex (MHC) molecule complexes on cell surfaces, we wish to know how such complexes interact with specific receptors to evoke the effector activities of mature T cells in the body, as well as regulate their growth, inactivation, or death. In particular, we want to understand in molecular detail the protein-protein interactions that turn recognition of antigen by T cells into signals guiding the normal survival and effector functions of these cells, how variations in these recognition and signaling events leads to desirable versus undesirable forms of immunity, and how we can manipulate these events to augment useful immune responses and inhibit damaging ones. Our studies currently focus on biochemical regulatory pathways that help T cells discriminate between self- and foreign peptide:MHC molecule complexes and on the explicit mathematical modeling of these signaling processes in eukaryotic cells. We previously reported our analysis of the molecular details of two novel regulatory pathways controlling early signaling by the T cell receptor (SHP-1 phosphatase dependent negative control and MAPK mediated positive control) and a full mathematical computer description of T cell receptor (TCR) signaling that incorporates these novel regulatory pathways. Last year, we published studies that address the issue of how naturally-occurring levels of intraclonal variation in protein expression affects individual T cell function and how co-regulation of diverse elements in a signaling network can contribute to minimizing the dispersive effects of stochastic protein expression on T cell function. Our data indicated that quantitative variations in CD8 expression would influence the antigen-sensitivity (EC50) of the entire population of T cells, whereas variation in SHP-1 would affect the proportion of cells showing any response, but not the antigen sensitivity (EC50) of the remaining proportion of activatable cells, and that variations in ERK, because it was in such excess, would have no measurable effects. All three predictions were borne out by studies using a novel method for simultaneously measuring protein expression in single cells and the signaling response of the same cells. Beyond this strong extension of the predictive power of our computational model, we also showed that opposing changes in a given cell in CD8 and SHP-1 expression eliminated the extremes of population distribution with respect to hypersensitivity or hyposensitivity of the T cells, providing a clue as to how the T cell manages to deal with noisy protein expression and show robust functional behavior over time. In the course of these studies, we also discovered that several hours after TCR stimulation most T cells begin to express the negative regulatory transcription factor Foxp3 that limits the extent and duration of cytokine production. A detailed analysis of the conditions leading to accumulation vs. extinction of Foxp3 expression in CD4+ T cells revealed that there is a major locus of control of expression of this protein at the post-transcriptional (protein) level. Our data reveal that Foxp3 is an intrinsically unstable protein highly dependent on molecular chaperones or partner protein binding for resistance to aggregation and degradation. Elevated temperature (fever), and a combination of strong TCR and CD28 stimulation that produces many client proteins for Hsp90 in the cytosol, limit access of Foxp3 to such stabilizing partners, resulting in its aggregation and degradation. Because Foxp3 positively regulates its own gene transcription, processes that limit accumulation of Foxp3 at the protein level have an amplified inhibitory effect by also reducing Foxp3 gene activity. Single cell studies showed a direct relationship in individual cells between the level of Foxp3 and cytokine gene responses. Thus, our results show that Foxp3 can serve as a cell autonomous regulator of effector function tuned to the host environment steady state conditions in which there is normal temperature, weak self ligand stimulation and low costimulation support Foxp3 expression that can interfere with autoreactive responses, whereas during infections, a combination of fever, strong foreign antigen stimulation, and strong costimulatory molecule expression suppress this regulatory loop by preventing Foxp3 protein accumulation, allowing effector responses that are needed by the host. Over the past year we have focused on using the level of CD5 expression to monitor the affinity of a specific TCR for self-MHC ligands. Using a variety of methods including analysis of partial TCR-associated zeta-chain phosphorylation, we found that we could readily classify T cells according to their self-ligand affinity by this means. We are now conducting experiments analyzing the role played by self-affinity in foreign antigen specific responses of T cells and in the proliferation of T cell in a lymphopenic environment. We are especially interested in understanding if the putative role of CD5 as a negative regulatory of T cell antigen reactivity is correct and if it is, whether CD5 levels are tuned only to prevent self-reactivity or if this molecule plays an additional role in regulating the polyclonality of responding T cell populations upon infection. We have also conducted experiments combining flow cytometric studies with intravital 2 photon imaging to assess the role of self-recognition in naive lymphocyte trafficking through lymph nodes. Our data reveal that we can quantitate clear differences in the length of CD4 T cell interactions with MHC class II+ vs. MHC class II - dendritic cells and that this is correlated with a difference in the time it takes naive CD4 T cells to transit through the lymph node of wild-type and MHC class II-deficient mice. These studies have also unexpectedly revealed a systematic difference in the length of time CD4 vs. CD8 T cells spend in a lymph node. These studies provide additional evidence for non-negligible interactions between the TCRs on naive T cells and ambient self-ligands in the normal host, interactions of sufficient magnitude to affect the dynamics of lymphoid populations with lymphoid tissue.